WO2016006782A1 - Composition favorisant la stabilité de stockage de cellules souches - Google Patents

Composition favorisant la stabilité de stockage de cellules souches Download PDF

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WO2016006782A1
WO2016006782A1 PCT/KR2015/000595 KR2015000595W WO2016006782A1 WO 2016006782 A1 WO2016006782 A1 WO 2016006782A1 KR 2015000595 W KR2015000595 W KR 2015000595W WO 2016006782 A1 WO2016006782 A1 WO 2016006782A1
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stem cells
composition
cell
serum
albumin
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Korean (ko)
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라정찬
우상규
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라정찬
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Priority to US15/126,231 priority Critical patent/US10172347B2/en
Priority to CN201580036349.3A priority patent/CN107072189B/zh
Priority to EP15818206.3A priority patent/EP3178318B1/fr
Priority to JP2016569967A priority patent/JP6741597B2/ja
Publication of WO2016006782A1 publication Critical patent/WO2016006782A1/fr

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
    • A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/16—Blood plasma; Blood serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/35—Fat tissue; Adipocytes; Stromal cells; Connective tissues
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/38—Albumins
    • A61K38/385—Serum albumin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/46—Ingredients of undetermined constitution or reaction products thereof, e.g. skin, bone, milk, cotton fibre, eggshell, oxgall or plant extracts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells

Definitions

  • the present invention relates to a composition capable of improving the storage stability of stem cells, and more particularly to a composition for improving the storage stability of stem cells containing serum or plasma.
  • Stem cells are cells that have the ability to self-replicate and differentiate into two or more cells.
  • Totipotent stem cells pluripotent stem cells, and multipotent stem cells ( multipotent stem cells).
  • Pluripotent stem cells are pluripotent cells that can develop into a complete individual. Cells up to 8 cell stages after fertilization of eggs and sperm have these properties. If you transplant it into a single, complete entity. Pluripotent stem cells are cells that can develop into a variety of cells and tissues derived from ectoderm, mesoderm, and endodermal layer. The inner cell mass inside the blastocyst appears 4-5 days after fertilization. It is called embryonic stem cells and differentiates into a variety of other tissue cells but does not form new life. Multipotent stem cells are stem cells that can only differentiate into cells specific to the tissues and organs that contain them.
  • Multipotent stem cells were first isolated from adult bone marrow (Y. Jiang et al., Nature , 418: 41, 2002) and subsequently identified in other adult tissues (CM Verfaillie et al., Trends Cell Biol. 12: 502, 2002).
  • stem cells in adult tissues such as bone marrow are very rare and these cells are difficult to culture without induction of differentiation, making them difficult to culture without specifically screened media. That is, it is very difficult to separate stem cells and preserve them in vitro.
  • adipose tissue has been found to be a new source of pluripotent stem cells (B. Cousin et al., BBRC, 301: 1016, 2003; A. Miranville et al., Circulation, 110: 349, 2004; S. Gronthos et al., J. Cell Physiol . 189: 54, 2001; MJ Seo et al., BBRC, 328: 258, 2005), and human adipose tissue obtained by liposuction (liposuction) contained undifferentiated cell populations.
  • liposuction liposuction
  • adipose tissue-derived cells have the ability to promote muscle regeneration and neurovascular differentiation. These adipose tissues have the advantage of being able to be extracted in large quantities, attracting attention as a source of new stem cells to supplement the existing disadvantages.
  • Stem cells which can be obtained in large quantities, are increasingly being used as cell therapeutics for injecting stem cells themselves for the medical field including treatment of intractable diseases and cosmetics and cosmetics.
  • Stem cells for use in cell therapy are not a problem when the patient can be treated immediately after the culture, but if the control of the stem cell treatment timing is necessary according to the condition of the patient, long-term preservation is required after the stem cell culture.
  • stable supply of stem cells is essential even when long distance transportation is required to the place where the stem cells are cultured.
  • the cell freezing method significantly lowers the cell viability at the time of thawing, and there are problems such as stem cell function from a biological point of view.
  • there is a disadvantage that the transport in the frozen state is more difficult than the refrigerated transport.
  • Conventional techniques for preserving stem cells in a refrigerated state currently include a technique for storing human adipose tissue-derived mesenchymal stem cells suspended in physiological saline under refrigerated conditions. It is hard to bet. In order to improve this, when the addition of sucrose or albumin shows a survival rate of 70% or more until 48 hours to 72 hours, it has been confirmed that the storage stability is improved (Korean Patent No. 2008-0103637). However, since the survival rate sharply decreases after 72 hours, it is necessary to develop a composition for enhancing storage stability that maintains the survival rate of stem cells included in the cell therapy in a refrigerated condition (4 ° C.) for a long time stably.
  • the present inventors have made diligent efforts to maintain high survival rate of stem cells even during long-term refrigeration, as a result of confirming that the survival rate of stem cells in the refrigerated state is maintained at least 9 days or more when containing serum or plasma. It was completed.
  • human serum is used as a medium composition for cell culture (Korean Patent No. 10-0394430), there is no disclosure about a composition for enhancing storage stability of stem cells containing human serum or plasma.
  • An object of the present invention to provide a composition for improving the stability of cold storage of stem cells containing 5 ⁇ 80% (v / v) content of serum or plasma.
  • Another object of the present invention to provide a cell therapy injection product containing the composition.
  • the present invention provides a composition for improving the stability of cold storage of stem cells containing a serum or plasma content of 5 ⁇ 80% (v / v).
  • the present invention also provides a cell therapy injection product containing the composition.
  • the control group used a preservative containing 0.3% albumin.
  • Figure 2 shows the survival rate of stem cells according to the retention time from 0 to 144 hours by refrigeration of 1.0 ⁇ 10 7 stem cells with preservatives containing autologous serum and 0.3% albumin at different concentrations (10, 20, 30%) It is a comparison.
  • the control group used a preservative containing 0.3% albumin.
  • Figure 3 shows the survival rate of stem cells according to the retention time from 0 to 144 hours by refrigerated 0.5 ⁇ 10 7 stem cells with preservatives containing different concentrations (10, 20, 30%) of taga serum and 0.3% albumin It is a comparison.
  • the control group used a preservative containing 0.3% albumin.
  • Figure 4 shows the survival rate of stem cells according to the retention time from 0 to 144 hours by refrigeration of 1.0 ⁇ 10 7 stem cells with preservatives containing different concentrations (10, 20, 30%) of taga serum and 0.3% albumin It is a comparison.
  • the control group used a preservative containing 0.3% albumin.
  • Figure 5 is a result of comparing the change in cell number, survival rate and cell size of the control group (saline / 0.3% albumin) of the stem cell # 1 and the composition experimental group (1-6) for enhancing the storage stability of each other conditions.
  • Figure 6 is a result of comparing the change in cell number, viability and cell size of the control group (saline / 0.3% albumin) of the stem cell # 2 and the composition experimental group (1-6) for enhancing the storage stability of each other conditions.
  • the stem cell preservative containing serum or plasma maintains the survival rate of the stem cells in the refrigerated state at least 95%, thereby improving the storage stability of the stem cells.
  • the stem cell preservative containing serum or plasma of the present invention can maintain a high survival rate of stem cells in a refrigerated state for 9 days or more, and is useful for stable supply of stem cells for cell therapy.
  • cell therapeutic injection product or “cell therapeutic agent” refers to a parenteral administration containing stem cells for injection of a defect of a tissue, that is, injected into or near a defect in the form of an injection to correct a defect. It means a pharmaceutical composition that can be.
  • excipient means a substance which is added to maintain the form of a liquid or the like, in addition to an active ingredient exhibiting pharmacological properties of the pharmaceutical composition, for the purpose of facilitating handling by a given dose and weight.
  • excipient for the liquid form, saline (Saline), Hartmann-D solution, PBS (Phosphate Buffered Saline), etc. are used, but it means a substance which may include various other compositions.
  • fat tissue-derived stem cell is an undifferentiated stem cell isolated from adipose tissue, and the separation method may be as follows.
  • Adipose tissue-derived stem cells may be isolated by culturing a suspension containing fat suspended in saline obtained by liposuction and then recovering the stem cell layer attached to the culture vessel by trypsin treatment or by scraping with a scraper.
  • stem cells of the present invention it can be obtained by the following method.
  • human adipose tissue obtained from abdominal fat by liposuction is isolated and washed with PBS, and then the tissue is chopped and digested with DMEM medium containing collagen degrading enzyme, washed with PBS and centrifuged. do. The supernatant was removed and the pellet was washed with PBS and then centrifuged again to remove suspended matter using a 100 ⁇ m mesh, followed by washing with PBS.
  • DMEM fetal bovine serum
  • keratinocyte-SFM FBS, NAC, Ascorbic Acid, Calcium, rEGF, insulin, bFGF and hydrocortisone
  • human adipose tissue-derived mesenchymal stem cells containing 10-50% (v / v) of autologous or other human serum and 0.3% albumin or a composition containing human plasma and 0.3% albumin The cells were suspended in the composition and analyzed for cell size, appearance, cell number, and viability over time in a refrigerated (4 ° C) state.
  • the present invention relates to a composition for enhancing the storage stability of stem cells containing 5 to 80% (v / v) serum or plasma.
  • the content of the serum is preferably 10 to 50% (v / v), the plasma content is preferably 5 to 50% (v / v), but is not limited thereto.
  • Serum of the present invention may be characterized in that the serum or plasma derived from mammals, including humans. It is also preferred to be allogeneically derived serum or plasma, and there is no limitation on the use of autologous or titer serum or plasma.
  • the refrigerated storage is preferably in the temperature range of 0.1 ⁇ 10 °C, but is not limited thereto.
  • the present invention may be further characterized by containing albumin and excipients
  • the content of the albumin is preferably 0.1 ⁇ 1% (v / v), it is 0.2 ⁇ 0.5% (v / v) More preferably, it is not limited thereto.
  • the excipient is preferably any one or more selected from the group consisting of saline, Hartman-D solution and PBS, but is not limited thereto.
  • V / v means that 1 to 80% of serum and 0.1 to 1% of albumin are added to the volume (volume percentage) of the stem cells suspended in saline solution so that the total dose (volume percentage) becomes 100%. do.
  • the concentration of the stem cells is preferably 1.0 ⁇ 10 5 ⁇ 1.0 ⁇ 10 9 cells / ml, more preferably 1.0 ⁇ 10 6 ⁇ 1.0 ⁇ 10 8 cells / ml, but is not limited thereto It is not.
  • the stem cells are preferably selected from the group consisting of fat, umbilical cord blood, bone marrow, muscle, placenta and skin, and most preferably those derived from adipose tissue, but are not limited thereto.
  • the stem cells may be characterized in that the stem cells derived from mammals, including humans.
  • the present invention relates to a cell therapeutic injection product containing a composition for enhancing the stability of cold storage of stem cells containing serum or plasma.
  • Injectable products according to the invention can be prepared in the form of filled injections, taking the amounts commonly known in the art, depending on the constitution and type of defect of the patient.
  • Injectable products according to the present invention can be used by injection in the region adjacent to the defect to be treated or defects, defects that can be corrected in this way are wrinkles, stretch marks, scars, skin depression, lip insufficiency, periodontal defects, Soft tissue defects, bone defects, burns, skin ulcers, etc., but is not limited thereto.
  • the cell therapeutic injection product may further contain a suspension, a dissolution aid, a stabilizer, a tonicity agent, a preservative, an adsorption agent, a surfactant, a diluent, a pH adjuster, a pain-free agent, a buffer, a sulfur-reducing agent, and an oxidation if necessary.
  • An inhibitor etc. can be added suitably.
  • Human adipose tissue obtained from abdominal fat by liposuction was isolated and washed with PBS. After cutting the tissue finely, the tissue was digested at 37 ° C. for 2 hours using DMEM media to which collagenase type 1 (1 mg / ml) was added. The collagenase treated tissues were washed with PBS, centrifuged at 1000 rpm for 5 minutes, the supernatant was removed, the pellet was washed with PBS and centrifuged at 1000 rpm for 5 minutes.
  • Example 2 Four different adipose stem cells isolated by the method of Example 1 were refrigerated at 4 ° C to analyze the survival rate of the stem cells used in the injectable product for a cold cell type therapeutic.
  • the four isolated adipose stem cells constituted the experimental group as shown in Table 1.
  • Stem cell control group containing 0.3% albumin
  • experimental group containing 0.3% albumin + 10% autologous serum experimental group containing 0.3% albumin + 10% taga serum
  • experimental group containing 0.3% albumin + 20% autologous serum 0.3
  • the survival rate was analyzed by including an experimental group containing% albumin + 20% taga serum, an experimental group containing 0.3% albumin + 30% autologous serum, and an experimental group containing 0.3% albumin + 30% taga serum.
  • pH is a numerical value of hydrogen ion concentration, and the general pH range is about 5.5 ⁇ 8.0 for saline, so the change of pH when albumin and human serum were added to saline was compared with PBS. .
  • Normal healthy pH is when cells, blood, and body fluids maintain weak alkalinity.
  • Thermo Scientific's Orion Star A111 was used to analyze the pH change by albumin and human serum included in the composition for enhancing stem cell storage stability. In addition, since pH is affected by the measurement temperature, it was measured at room temperature in the range of 20 ⁇ 24 °C.
  • Example 3 the stem cell control suspended in saline containing 0.3% albumin and the saline containing 10, 20, 30% autologous human serum and 0.3% albumin, respectively. Viability analysis was performed with the stem cell experimental group and the stem cell experimental group suspended in saline containing 10, 20, and 30% taga human serum and 0.3% albumin, respectively.
  • Adipocytes isolated by the method of Example 1 were washed with PBS, suspended in saline containing 0.3% albumin and added with 10, 20, and 30% autologous and other human serum, respectively. It consisted of 2 control group and the experimental group.
  • Stem cells in each experimental group were filled into 3cc syringes at a concentration of 1.0 ⁇ 10 7 cells / ml, then refrigerated at 4 ° C., and then injected into a cold cell-type cell therapeutic product after 0, 24, 48, 72, 96, 120 and 144 hours.
  • the size and shape of stem cells used in the study were analyzed.
  • Cell 1 was suspended in saline containing 0.5 ⁇ 10 7 stem cells in saline containing 0.3% albumin and saline containing 10, 20, 30% autologous serum / 0.3% albumin, respectively.
  • Three stem cell experimental groups were prepared.
  • Cell 2 is a control group in which 1.0 ⁇ 10 7 stem cells are suspended in saline containing 0.3% albumin, and stem cells suspended in saline containing 10, 20, 30% autologous serum / 0.3% albumin, respectively. Three experimental groups were prepared.
  • control group of cell 1, the control group of 3 and the control group of cell 2, and the control group of 3 were all filled with stem cells in a 3cc syringe, and then refrigerated at 4 ° C and after 0, 24, 48, 72, 96, 120 and 144 hours.
  • the size, appearance and total cell number of were analyzed.
  • the cell number and size of stem cells were analyzed using Invitrogen's Countess TM Automated Cell Counter, and the characterization was visually confirmed.
  • Table 3 cell number of cells 1
  • Table 4 size of cells 1
  • Table 5 cell numbers of cells 2
  • Table 6 size of cells 2).
  • the change in cell size of the control group was reduced by about 15-25%, whereas the change in cell size of the experimental group containing autologous human serum was not significantly changed by about 3-10%.
  • no change was observed in the appearance of the cells in the control group and the experimental group, and the total number of cells was not different between the control group and the experimental group.
  • Cell 3 was a control group in which 0.5 ⁇ 10 7 stem cells were suspended in saline containing 0.3% albumin and saline containing 10, 20, 30% taga serum / 0.3% albumin, respectively. Three stem cell experimental groups were prepared.
  • Cell 4 was suspended in saline containing 1.0 ⁇ 10 7 stem cells in saline containing 0.3% albumin and saline containing 10, 20, 30% taga serum / 0.3% albumin, respectively.
  • Three stem cell experimental groups were prepared.
  • the control group of cell 3, the control group of 3 and the control group of cell 4, and the control group of cell 4 all filled the stem cells in a 3cc syringe, and then stored at 4 ° C and stored at 0, 24, 48, 72, 96, 120 and 144 hours.
  • the size, appearance and total cell number of were analyzed.
  • the cell number and size of stem cells were analyzed using Invitrogen's Countess TM Automated Cell Counter, and the characterization was visually confirmed.
  • Table 7 cell number of cells 3
  • Table 8 size of cells 3
  • Table 9 cell numbers of cells 4
  • Table 10 size of cells 4
  • the change in cell size of the control group was reduced by about 20%, whereas the change in cell size of the experimental group containing other human serum was not significantly changed by about 3 to 10%.
  • no change was observed in the appearance of the cells in the control group and the experimental group, and the total number of cells was not different between the control group and the experimental group.
  • the isolated adipose stem cells were washed with PBS, suspended in saline containing 0.3% albumin, and added with 10, 20, and 30% autologous and other human serum, respectively. .
  • Stem cells in each experimental group were filled into 3cc syringes at a concentration of 1.0 ⁇ 10 7 cells / ml, then refrigerated at 4 ° C., and then injected into a cold cell-type cell therapeutic product after 0, 24, 48, 72, 96, 120 and 144 hours.
  • the survival rate of stem cells used for the analysis was analyzed. Stem cell viability was measured using a 1: 1 1: 1 mixture of cells and trypan blue solution using Invitrogen's Countess TM Automated Cell Counter.
  • the composition containing human serum showed high survival rate without significant change in cell number compared to the control group containing only 0.3% albumin.
  • the experimental group 3 which is 30% serum-containing stem cells, maintained a very high cell viability of 98% or more until 144 hours, and it was confirmed that even in the 10-20% serum-containing experimental group, the survival rate was 90% or more until 72 hours later.
  • the effect of survival rate according to the retention time of cells there was no difference in the effect of survival rate according to the retention time of cells between the autologous serum test groups (FIGS. 1 and 2) and the taga serum test groups (FIGS. 3 and 4).
  • SA Saline + 0.3% Albumine
  • PRP plasma
  • Example 2 Two different adipose stem cells separated by the method of Example 1 were refrigerated at 4 ° C to analyze the survival rate of the stem cells used in the injectable product for a refrigerated cell therapy product.
  • 4.9 ⁇ 10 8 cells (1.0 ⁇ 10 7 cells / ml 49 preparations) were prepared for each cell. Thaw two passages (6.0 ⁇ 10 6 cells in five T175 flasks) and culture (4.9 ⁇ 10 7 cells in 49 T175 flasks in three passages), then recover the fourth passage (4.9 ⁇ 10 8 cells) and then filled and tested.
  • the cells recovered in the fourth passage were divided into 7 tubes with 7.0 ⁇ 10 7 cells, mixed with 7 ml of 7 preservatives in Table 11, and then hourly (0, 1, 2, 3, 5, 7, 9 per preservative). 1) was filled into 7 3cc syringes. Day 0 samples were immediately measured cell number, viability, and cell size, and the rest of the samples were stored refrigerated for 1, 2, 3, 5, 7, 9 days, and then cell number, viability, and cell size using Cedex. Measured.
  • Plasma was injected into a 20cc syringe with 2cc of anticoagulant, and after blood collection, the plasma and erythrocytes were firstly separated by Dr. PRP kit, followed by centrifugation at 3200rpm for 6 minutes to condense platelets and secondary separation. After the second separation, in the state where PRP (platelet-containing plasma) and PPP (platelet-free plasma) were separated, 4 ml of PPP in the upper layer was slowly removed with a 10cc syringe, and the remaining PRP 4ml was mixed well.
  • Example 2-2 the pH of the excipients of the composition for enhancing stem cell storage stability of each experimental group was analyzed.
  • Thermo Scientific's Orion Star A111 was used to analyze the pH change by albumin and human serum included in the composition for enhancing stem cell storage stability.
  • NaHCO 3 sodium bicarbonate
  • Adipocytes isolated by the method of Example 1 were washed with PBS, and then cells were prepared by the method of Example 5-1. As shown in Table 11, two kinds of stem cells were composed of a control group and six experimental groups, and cell number, viability and cell size were examined up to 9 days.
  • control group SA: Saline + 0.3% albumin
  • experimental group containing 10% plasma or 10% serum was up to 50% higher than the control group.
  • the effect of increasing the survival rate of more than% was confirmed (Figs. 5 and 6).
  • Serum concentration of 50% showed the highest cell viability, but it was confirmed that the experimental group containing 30% serum also showed cell viability of 90% or more until 9 days (Figs. 5 and 6).
  • stem cells when preserving stem cells using a preservative including serum or plasma, it was confirmed that the stem cells can be stored for a long time without change in cell number, viability and cell size by enhancing the storage stability of stem cells.
  • the storage stability of stem cells was compared by increasing the concentration of serum as a method to enhance the effect of the storage stabilizer.
  • serum / SA serum + 0.3% albumin
  • the survival rate was increased after increasing the serum concentration to 10, 30, and 50%.
  • the stabilizer containing 50% serum showed the highest survival rate.
  • the stabilizing agent containing 10% serum maintained a survival rate of 80% or more for 7 days, and the stabilizer containing 30% serum showed a very high survival rate of 90% or more for 9 days (FIG. 10). This indicates that serum plays a large role in maintaining the survival rate of refrigerated cells.
  • composition for enhancing the storage stability of stem cells according to the present invention can maintain a survival rate of at least 90% or more without changing the properties, number and size of the stem cells for more than 9 days, long-term transport and effect of stem cells for cell therapy It is also useful for the manufacture of superior cell therapeutic injection products.

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  • Organic Chemistry (AREA)
  • Physiology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Rheumatology (AREA)
  • General Engineering & Computer Science (AREA)
  • Botany (AREA)
  • Inorganic Chemistry (AREA)
  • Dermatology (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicinal Preparation (AREA)

Abstract

La présente invention concerne une composition capable de favoriser la stabilité de stockage de cellules souches. Plus spécifiquement, la présente invention concerne une composition qui contient un sérum ou un plasma favorisant la stabilité de stockage au froid de cellules souches. La composition favorisant la stabilité au stockage de cellules souches selon la présente invention peut maintenir un taux de survie supérieur à 90 % pendant au moins 9 jours sans changement des propriétés, du nombre ou de la taille des cellules souches, et est ainsi utile pour le transport de longue durée de cellules souches pour la thérapie cellulaire et la préparation de produits thérapeutiques cellulaires d'injection ayant un effet excellent.
PCT/KR2015/000595 2014-07-08 2015-01-21 Composition favorisant la stabilité de stockage de cellules souches WO2016006782A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
US15/126,231 US10172347B2 (en) 2014-07-08 2015-01-21 Composition for improving stability of stem cells
CN201580036349.3A CN107072189B (zh) 2014-07-08 2015-01-21 用于改善干细胞稳定性的组合物
EP15818206.3A EP3178318B1 (fr) 2014-07-08 2015-01-21 Composition favorisant la stabilité de stockage de cellules souches
JP2016569967A JP6741597B2 (ja) 2014-07-08 2015-01-21 幹細胞の安定性増進用組成物

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KR20140085020 2014-07-08
KR10-2014-0085020 2014-07-08

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WO2016006782A1 true WO2016006782A1 (fr) 2016-01-14

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EP (1) EP3178318B1 (fr)
JP (1) JP6741597B2 (fr)
KR (1) KR101597604B1 (fr)
CN (1) CN107072189B (fr)
WO (1) WO2016006782A1 (fr)

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EP3581644A4 (fr) * 2017-02-10 2020-01-29 FUJIFILM Corporation Conservateur de cellules souches humaines, suspension de cellules souches humaines, et procédé de conservation de cellules souches humaines

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US20210008120A1 (en) * 2018-03-13 2021-01-14 Oxacell Ag Cell products with improved stability and uses thereof
JP6594578B1 (ja) * 2018-04-25 2019-10-23 セルトラスト・アニマル・セラピューティクス株式会社 細胞の保存方法および細胞懸濁液
CN108849857A (zh) * 2018-07-20 2018-11-23 吉林济惠生物科技有限公司 一种脐带间充质干细胞的运输保护液

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3581644A4 (fr) * 2017-02-10 2020-01-29 FUJIFILM Corporation Conservateur de cellules souches humaines, suspension de cellules souches humaines, et procédé de conservation de cellules souches humaines

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US10172347B2 (en) 2019-01-08
JP2017521378A (ja) 2017-08-03
US20170105406A1 (en) 2017-04-20
EP3178318A1 (fr) 2017-06-14
JP6741597B2 (ja) 2020-08-19
EP3178318B1 (fr) 2020-08-26
KR101597604B1 (ko) 2016-02-26
CN107072189B (zh) 2021-07-20
KR20160006127A (ko) 2016-01-18
CN107072189A (zh) 2017-08-18
EP3178318A4 (fr) 2017-12-13

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